Chlamydia are Gram-negative bacterial pathogens that infect a wide range of hosts and cause various diseases, including preventable blindness in developing countries, sexually transmitted disease and pneumonia. Chlamydia replicate intracellularly in a membrane-bound compartment that potentially serves as a protector shield against immune surveillance but also acts as a "platform" to exchange factors with the host cell. Despite the primary importance of Chlamydia as a human pathogen, little is known about the bacterial and host factors involved in the infection process. This paucity of knowledge is mainly due to the fact that Chlamydia is not a genetically tractable organism and to the difficulty of conducting genetic approaches in the mammalian host. We propose to conduct a genome-wide siRNA screen in order to identify and characterize human host factors involved in post-invasion events of Chlamydia trachomatis infection. We have demonstrated the feasibility of the approach on a subset of the genome and we propose to extend the approach to the entire human genome (Aim#1). Since RNAi treatment has been shown to be associated with the unintended silencing of genes displaying limited sequence homology with the targeted gene, we will perform a systematic validation procedure of the candidates to unambiguously establish a functional relationship between the knock-down of the targeted gene and the observed phenotype (Aim#2). Finally, we will conduct secondary assays to identify and characterize Chlamydia specific candidates involved in post-invasion events of C. trachomatis intracellular development (Aim#3). Overall, this developmental proposal is likely to provide new insights in Chlamydia pathogenesis and to generate the required preliminary results for future funding application(s) related to the characterization of known and/or novel pathway(s) involved in C. trachomatis infection. PUBLIC HEALTH RELEVANCE: Chlamydia are obligate intracellular bacterial pathogens responsible for various diseases such as trachoma, sexually transmitted disease and pneumoniae. We study how Chlamydia hijack cellular components to successfully replicate and disseminate inside the host. We focus on dissecting the cellular mechanism(s) invloved in the infection process by identifying the host factors required for Chlamydia development. The identification of these factors may help design new therapeutic treatments.